METABOLISM of COUMAPHOS* in LARVAE of the CATTLE TICK BOOPHILUSMIOROPLUS by W

Home , Coumaphos

METABOLISM OF COUMAPHOS* IN LARVAE OF THE CATTLE TICK BOOPHILUSMIOROPLUS By W. J. ROULSTON,t C. A. SCHUNTNER,t and H. J. SCHNITZERLINGt

[ManU8cript received June 7, 1965]

Summwry

[82P]Coumaphos, a phosphorothionate, was rapidly absorbed and metabolized by cattle tick larvae dipped in aqueous suspensions of the acaricide. One metabolite was shown by chromatographic, spectroscopic, and cholinesterase inhibition evidence to be the oxygen analogue, a more potent in vitro cholinesterase inhibitor than the parent compound. Other water-soluble~ metabolites were only tentatively identified. Sublethal doses were metabolized mainly to water-soluble materials and a small quantity of oxygen analogue whereas lethal doses, producing complete mortality in 2-3 hr, yielded almost equal amounts of the oxygen analogue and water-soluble materials. In vivo inhibition of cholinesterase in treated larvae was dose-dependent and preceded death. 1. INTRODUCTION Control of the Australian cattle tick, Boophilu8 microplu8 (Canestrini), can be obtained with a number of chemicals belonging to the organic phosphorus group (Roulston and Wilson 1965). As a first step towards understanding the mode of action of these chemicals a study was initiated to establish both qualitatively and quantitatively the metabolism of a candidate organic phosphorus acaricide in cattle ticks. Coumaphos (I) was selected as the candidate compound because of its high level of effectiveness under field conditions and ready availability. The study took on greater significance when a strain of ticks resistant to a number of organic phosphorus compounds was found in Queensland (Shaw and Malcolm 1964).

II. MATERIALS AND METHODS (a) Ohemicals The [32P]coumaphos § was shown to be radiochemically pure by paper chromatography in three systems and spectroscopically pure by infrared analysis. The specific activity was 4·3 mc/g at the commencement of experiments. Non radioactive coumaphos§ (m.p. 93°C) was prepared from a sample of 98% coumaphos by repeated recrystallization from n-hexane.

* A common name recommended by British Standard 1831 : 1961 for 3-chloro-4-methyl- 7-coumarinyl diethyl phosphorothionate, which is also known as Bayer 21/199, Co-Ral, and Asuntol. t Division of Entomology, CSIRO, Veterinary Parasitology Laboratory, Yeerongpilly, Qld. ~ As indicated in Table 1. § Supplied by Farbenfabriken Bayer AG. through the courtesy of Dr. E. Endrajat.

AU8t. J. Biol. Sci., 1966, 19, 619-33 620 W. J. ROULSTON, C. A. SCHUNTER, AND H. J. SCHNITZERLING

The oxygen analogue of coumaphos (II, 3-chloro-4-methyl-7 -coumarinyl diethyl phosphate) was prepared by bromine oxidation of purified coumaphos in aqueous ethanol solution. After extraction and recrystallization a white crystalline solid was obtained, m.p. 71-72°C (Found: C, 49·0; H, 5·0; CI, 10·4; P, 9·0. C14H16CI06P requires C, 48·5; H, 4·6; CI, 10·2; P, 8·9%). The [32P]-oxygen analogue was prepared similarly.

CzH:,o" -CX°,,-c~o CZH50, -;:?o p-o I I 'p-o-0:°,,- ~ I C /,\ ~C /,\ I CZH50 S ~ C;/ "--..C1 CzHsO ° ~ C~C"--..C1 I I CHJ CH3 (I) (II)

Technical diethyl phosphoric, * diethyl thiophosphoric, * and diethyl dithio phosphoric* acids were purified by column chromatography on silicic acid by the method of Schnitzerling and Schuntner (1965).

(b) Treatment The suspensions for dipping larvae were formed by dissolving [32P]coumaphos in ethanol containing Triton X-IOO in such proportions to give, on dilution with water, suspensions containing 1 % ethanol and 0·02% Triton X-IOO. The suspensions were prepared just prior to use by adding 1 ml of alcoholic solution to 100 ml of distilled water. Larvae, approximately 14 days old, were dipped for 4 min, using the ratio of 1 g larvae to 5 ml of suspension, after which they were dried and incubated at 30°C and high humidity. Death of larvae was assumed when no leg movements could be observed and the larval mass separated freely on rotating the container.

(c) Extraction of Radioactive Compounds All samples of larvae for analysis were washed free of external radioactive compounds by shaking with a 1 : 1 mixture of methanol and acetone. Internal radioactive compounds were extracted from small samples of larvae by homogenizing in an all-glass homogenizer with methanol, evaporating the supernatants to small volumes, and spotting on paper for preliminary chromatographic separations. Large samples of larvae were extracted with methanol in a reciprocating steel ball mill and the supernatants evaporated to the aqueous stage for liquid-liquid extraction of radioactive compounds. Although methanol was the most effective solvent tested for extracting the radioactive compounds, the larval residues after extraction and centrifugation still contained up to 20% of the original internal radioactivity. Most of this was readily extracted by warming with IN methanolic potassium hydroxide for a few minutes, and then slightly acidifying the extract with ION hydrochloric acid to prevent alkaline hydrolysis of radioactive compounds. The stability of coumaphos to the alkaline extraction method was proved by the quantitative recovery of [32P]coumaphos

* Supplied by Farbenfabriken Bayer AG. through the courtesy of Dr. E. Endrajat. METABOLISM OF COUMAPHOS IN CATTLE TICKS 621 added to the methanol-extracted residues of untreated larvae just prior to alkaline extraction, although reversible ring opening may have occurred (Kane et al. 1960).

(d) Separation of Metabolites The metabolites were separated by two different methods. The first depended entirely on paper chromatography and the second method used liquid-liquid solvent extraction to rid extracts of interfering compounds and effect separations into classes of compounds before using paper chromatography. (i) Ohromatographic Separation Five ascending chromatographic systems were used at 27°C for the separation and subsequent clean up of fractions. The solvent systems were generally modifications of systems previously reported. The components of these systems are listed in the following tabulation: System Components Reference Silicone 550; water-ethanol-chloroform Kaplanis, Hopkins, and Treiber (1959) 2 Carbowax; water-ethanol-chloroform Kaplanis, Hopkins, and Treiber (1959) 3 Silicone 550; methyl cyanide-water Chamberlain, Gotterdam, and Hopkins (1960) 4 Whatman No.1 paper; isopropyl Plapp and Casida (1958) alcohol-ammonium hydroxide 5 Whatman No.1 paper; 5% methanol This paper in n-hexane (v/v) Chromatography of all methanol and alkaline methanol extracts was first performed in system 1 as this had the greatest capacity for biological extractives. System 2 provided further clean up and confirmation of the identity of coumaphos and the oxygen analogue while system 3 was capable of resolving the water-soluble metabolites. System 4 provided additional evidence for identity of the water-soluble metabolites, while system 5 was used in the final clean up of extracted oxygen analogue metabolite. Whatman No. 31 E.T. paper was used for initial chromatograms in 3-cm wide strips for small-sample extracts (0·5-1 g larvae) or in sheet form for large samples (lOO g). When the load of extractives was sufficiently reduced to allow separations, Whatman No.1 paper in 3-cm wide strips was used. Chromatograms were scanned for radioactivity and the fractions eluted with methyl cyanide for solvent-soluble fractions or with methanol (containing 2% hydrochloric acid where elution was difficult) for water-soluble fractions. (ii) Liquid-Liquid Separation Methyl cyanide extracts (Table 1) were chromatographed in systems 1 and 2 and the ether and amyl alcohol extracts in system 3. (e) Oounting and Scanning Liquid extracts were counted by a scaler with an annular type Geiger-Muller detector. The activity on chromatograms was automatically recorded after scanning by a thin end-window Geiger-Muller detector. 622 W. J. ROULSTON, C. A. SCHUNTER, AND H. J. SCHNITZERLING

(f) Spectroscopy Ultraviolet spectroscopy of reference and radioactive materials and larval extractives was carried out in n-hexane solution. Infrared spectra were obtained using the potassium bromide pellet technique.

TABLE 1

OUTLINE OF THE LIQUID-LIQUID SEPARATION METHOD

Homogenized Larvae

Extracted four times with methanol (4 : I v/w). Evaporated to aqueous stage and brought to 1 : 1 v/v with methanol. Extracted four times with benzene (1 : 1 v/v)

Benzene phase Aqueous phase

Taken to dryness. Residue Acidified to IN with dissolved in n·hexane. HCl. Extracted five Extracted four times with times with diethyl methyl cyanide (saturated ether (1 : 1 v/v) with n·hexane--l : 1 v/v)

I I I Methyl cyanide n·Hexane Diethyl ether Aqueous I phase phase phase phase I Paper chroma· Extracted four tographyof times with I residue from amyl alcohol I this phase (1 : 1 v/v) I I I I I Coumaphos Oxygen Small Amyl alcohol Aqueous I analogue amount phase phase of coumaphos of polar I material

I \ v v J I Solvent·soluble Water-soluble

(g) Oholinesterase Activity and Inhibition The Warburg manometric method was used throughout. Bovine red blood cells were used as a source of enzyme activity (Radeleff and Woodard 1956) in the in vitro tests of inhibition of cholinesterase activity by [32P]-labelled metabolites, METABOLISM OF COUMAPHOS IN CATTLE TICKS 623

inactive reference compounds, and larval extractives. For all assays dilution of the red blood cells was adjusted so that 0·2 ml, on addition to the enzyme medium, produced an evolution of 100 1-1-1 of carbon dioxide in 1 hr. The final volume of the enzyme medium was always 3 ml. The final concentrations of the medium constituents were acetylcholine 0·0066M, sodium bicarbonate 0'025M, and sodium chloride 0·5M. Control reaction solutions containing all constituents except inhibitor, and blank reaction solutions containing all constituents except acetylcholine, were run for all assays. The flasks were gassed with a mixture containing 95% nitrogen and 5% carbon dioxide.

TABLE 2 RF VALUES FOR NON-RADIOACTIVE COUMAPHOS AND POSSIBLE METABOLITES AS PURE COMPOUNDS DETECTED WITH ACID PERMANGANATE OR HANES AND ISHERWOOD REAGENT FOR PHOSPHATES

RF Values Solute System 1 System 2 System 3 System 4

Coumaphos 0·16 0·87 0·99 0·86

Oxygen analogue 0·74 0·39 0·99 0·76

Diethyl phosphoric acid 0·96 0·00 0·19 0·65

Diethyl thiophosphoric acid 0·98 0·00 0·39 0·68

Diethyl phosphate (potassium salt) 0·94 0·00 0·16 0·62

Diethyl thiophosphate (potassium salt) 0·98 0·05 0·38 0·74

All the compounds tested with the exception of coumaphos were sufficiently water-soluble to give the required concentrations. It was not possible to increase the final coumaphos concentration beyond 1· 5 X 1O-4M without crystallization taking place, even in the presence of such relatively high final concentrations as 1 % acetone or 1 % ethanol and O· 05 % Triton X-100. The reference compounds diethyl phosphoric and thiophosphoric acids were neutralized to pH 7 with 0 ·IN potassium hydroxide immediately prior to inhibition assay. Mter 15 min gassing and 15 min temperature equilibration at 37°C, the taps on the manometers were turned off and the side-arm contents containing the inhibitor or blank solutions immediately tipped into the main flask contents. The concentration of acetylcholine at the time of inhibitor addition was O' 0060M. The percentage inhibition was calculated from the total volumes of carbon dioxide evolved from control and inhibitor reaction solutions after a reaction period of 1 hr. In the in vivo tests for the determination of cholinesterase inhibition, larvae were treated with various concentrations of coumaphos suspensions prepared as previously described, and larvae to be used as controls were treated with solutions without coumaphos. At various intervals control and treated O· 5-g samples of larvae were homogenized in 5 ml of an ice-cold solution containing O· 066M acetylcholine which should have been given protection against any free inhibitor (van Asperen 1960). The homogenate was centrifuged at 1000 r.p.m. for 3 min and 0·5 ml of the 624 W. J. ROULSTON, C. A. SCHUNTER, AND H. J. SCHNITZERLING supernatant was added to a Warburg flask already containing 2·5 ml of the homogenizing fluid without acetylcholine (van Asperen 1960). The flasks were gassed for 5 min followed by 5 min temperature equilibration at 37°C. The time

CHROMATOGRAPHIC SEPARA nON LIQUID-LIQUID SEPARATION

7 ..

SOLVENT -SOLUBLE SOLVENT -SOLUBLE

'< 1;; :'S l? '"w ~

:r:o ~ .-\ ::;: .\.:-., ::0 f, o U )(...... --~-...... =0 :> :5 8 WATER-SOLUBLE WATER-SOLUBLE "::l ~-----< /~

! ! , ! , ,!! I I ,! I! 10 12 14 16 18 20 22 24 0 10 12 14 16 18 HR AFTER DIPPING

Fig. i.-Penetration and metabolism of [32P]coumaphos in larval ticks after treatment in 0·0002% coumaphos suspension for 4 min as indicated by the chromatographic and liquid-liquid separation methods. Ratio of volume of suspension to weight of larvae 2·5 : 1 in the former and 5 : 1 in the latter method. ~ Coumaphos "outside" ) 6. Ether residue 1 Amyl alcohol residue • Coumaphos "inside" o Solvent • Aqueous residue Water X Oxygen analogue of soluble 8 Alkali·labile residue fsoluble coumaphos • Total water·soluble o Hexane residue materials from the commencement of homogenization to the commencement of reaction measurement was 30±1 min. The percentage inhibition was calculated after 20 min incubation at 37°C. Mortality counts were made on treated and control larvae at approximately the same time as samples were taken for the determination of cholinesterase inhibition. Mortality was determined on the basis of no discernible leg movements. METABOLISM OF COUMAPHOS IN CATTLE TICKS 625

III. RESULTS (a) Penetration and Metabolism Coumaphos and some of the metabolites were tentatively identified by com parison of their RF values (Table 2) with those of reference compounds chromato graphed in four solvent systems. It was found that [32P]coumapp.os penetrated

CHROMATOGRAPHIC SEPARATION LIQUID-LIQUID SEPARATION 70 .. 60 SOL VENT-SOLUBLE SOL VENT-SOLUBLE

w 30 « > :5'" c.!) 20 ~ ~ o'" 10 ~. J: ~ L _____/ - ~ __x ~ 0 o u ,,: :; WATER-SOLUBLE WATER-SOLUBLE ~ b :i..

o I I I o 2: o HR AFTER DIPPING

Fig. 2.-Penetration and metabolism of [32P]coumaphos in larval ticks after dipping in 0·002% coumaphos suspension for 4 min as indicated by the chromatographic and liquid-liquid separation methods. Ratio of volume of suspension to weight of larvae 5 : 1 in both methods.

.... Coumaphos "outside" t:, Ether residue • Coumaphos "inside" • Aqueous residue X Oxygen analogue of coumaphos ~ Total water-soluble materials larval ticks rapidly and that there was deteetable metabolism within 1 hr whether the acaricide was applied as a low dose (Fig. 1) or a high dose (Fig. 2). With the low dose of coumaphos there was approximately 60% larval mortality after 25 hr, but with the high dose all larvae were dead after 2-3 hr. 626 W .•T. ROULSTON, C. A. SCHUNTER, AND H. J. SCHNITZERLING

(b) Characterization of Metabolites (i) Solvent-soluble Metabolites The main solvent-soluble metabolite behaved as the reference oxygen analogue on rechromatography in systems 2 and 3. In Figures 3A and 3B are shown two typical separations in solvent system 1 of methanol extracts of larvae treated with

~ (1) ~--=.--'------,-----c------"'=- -=f-"- ~t)- o 0·5

o ~ D~ I ~I ~

EL" ~ I ~I' o ~ RF

Fig. 3.-Radiochromatograms of extracts of larvae treated with 0·0002% coumaphos (A) and 0·002% coumaphos (B) in solvent system 1. 0, D, E, rechromatograms of fraction (ii) from extracts of larvae treated with 0·002 % coumaphos in solvent systems 1, 2, and 3, respectively. Fraction (i) shown to be coumaphos, fraction (ii) oxygen analogue of coumaphos, and fraction (iii) water-soluble metabolites.

0·0002 and 0·002 % coumaphos. Both were sampled 3 hr after treatment of larvae and appeared qualitatively similar. Fraction (i) was coumaphos and fraction (ii) the oxygen analogue. Figures 3C, 3D, and 3E show rechromatography of fraction (ii) of Figure 3B in systems 1, 2, and 3. This confirmed that fraction (ii) was substantially METABOLISM OF COUMAPHOS IN CATTLE TICKS 627 a single compound. Fraction (ii) from sublethally dosed larvae behaved similarly. Fraction (iii) water-soluble materials from Figures 3A and 3B gave different resolutions when rechromatographed in system 3 as shown in Table 3. Clean up of fraction (ii) was achieved by successive chromatography and elution of the required fractions using systems 1, 3, 2, and 5. The fraction was freed of Carbowax from system 2 by partitioning between hexane and water. The hexane solution was extracted with dilute sodium bicarbonate solution to free the fraction from acidic extractives before the final clean-up in system 5.

TABLE 3 RF VALUES AND TENTATIVE IDENTIFICATION OF METABOLITE FRACTIONS

Rp Values Tentative Fraction Identification System 1 System 2 System 3 System 4 of Metabolite

Apparent oxygen 0·64-0·74 0·35-0·50 1·0 0,77-0,82 Oxygen analogue of analogue coumaphos

Water-soluble [fraction 0·84-0·94 0·00-0·02 0·18-0·20 0·67-0·71 Diethyl phosphoric (iii), Figs. 3A and 3B] acid

Water-soluble [fraction 0·84-0·94 0·00-0·07 0·37-0·52 0·71-0·73 Diethyl thio- (iii), Figs. 3A and 3B] phosphoric acid

Water-soluble (lethal 0·84-0·94 0·00-0·04 0·84-0·86 0·81 Intermediate dose) [fraction (iii), metabolite (?) Fig.3B] Minor metabolites 0·84-0·94 0·20 }DesethYI (water-soluble) 0·14 compounds (?) 0·00 Orthophosphoric acid

The ultraviolet and infrared spectra of purified fraction (ii) and reference oxygen analogue shown in Figures 4 and 5, respectively, are, with the exception of a limited region of the infrared spectra, completely in agreement. This discrepancy is due to the presence of a very small amount of .larval extractive which produced a weak undifferentiated absorption band in the region 1300-1000 cm-I .

(ii) Water-soluble Metabolites When fraction (iii) (see Fig. 3) from chromatograms of larvae treated with' sublethal doses was rechromatographed in system 3, two major components could be tentatively identified as diethyl thiophosphoric acid and diethyl phosphoric acid_ Rechromatography of these fractions in system 4 gave additional support to this identification. Fraction (iii) from lethally dosed larvae gave a mixture containing some of the above metabolites and an additional compound corresponding to the solvent-soluble polar material (Table 1). Attempts to obtain spectroscopically pure water-soluble metabolites were not successful due to the difficulty of separation from extractives and the volatility of the metabolites. 628 W. J. ROULSTON, C. A. SCHUNTER, AND H. J. SCHNITZERLING

0·6

0·4

0·2 ....> v; Z u.J C\ --' 0 <{ !,,! o5: 0·6

0·4

0·2

ci I ! I ! I I I 350 330 310 290 270 250 230 210 WAVELENGTH (m!,)

Fig. 4.-Ultraviolet absorption spectra of: A, fraction (ii); B, reference oxygen analogue of coumaphos.

1·5 1·0 0·8 0·6

0·4

0·2

0 1·5 1·0 0·8 0·6

0-4. 0·2~ I I 1 I 1111 I I II h I ! ! 2000 1800 1600 1400 1200 1000 800 600 fREQUENCY (eM"] )

Fig. 5.-Infrared spectra of: A, fraction (ii); B, reference oxygen analogue of coumaphos. iU. METABOLISM OF COUMAPHOS IN CATTLE TICKS 629

(c) Metabolism in Larvae Treated with Low and High Doses of Coumaphos In the experiments with a high dose rate of coumaphos the radioactive com pounds in the aqueous residues from the benzene extraction (water solubles) could be almost completely extracted with ether. However, water-soluble metabolites produced from the lower dose rate were only partly extractable with ether, most of the radioactivity being extracted with isoamyl alcohol. This variation in extractability with dose rate was paralleled in the chromatographic separations, where qualitative differences appeared in the polar fractions from paper chromatograms of methanol extracts of lethally and sublethally dosed larvae when chromatographed in systems 2

TABLE 4 INHIBITION OF CHOLINESTERASE BY FRACTION (ii) AND REFERENCE COMPOUNDS AND THEIR Iso VALUES

Inhibition by Inhibitor Inhibition by Inhibition by Oxygen Analogue Concentration Fraction (ii) Coumaphos of Coumaphos (M) (%) (%) (%)

2·0x 10-7 - 15 (av. 2 values) - 6·0 X 10-7 34 (av. 6 values) - - 1·0xl0-6 - 34 (av. 2 values) - 1·lxl0-6 - 51 (av. 3 values) - 1·2 X 10-6 55 (av. 6 values) - - 2·4 X 10-6 68 (av. 6. values) - - 5·0 X 10-6 - 77 (av. 2 values) - 2·4xl0-5 - - 5·0x 10-5 - - 24 (av.4 1·0x 10-4 ll} - - 38 values) 1·5xl0-4 - - 43

150 (M) 1·lxl0-6 1·2 X 10-6 1·8 X 10-4* --- * Extrapolated value. and 3 (Figs. 1 and 2). The results from low and high doses of coumaphos also differed in the ratios of the methanol-extracted, water-soluble metabolites to the amount of oxygen analogue. Thus after approximately 1 hr this ratio was 3·6 in the chromato graphic separations (low dose, Fig. 1) compared to 1· 3 and 0·8 in the chromatographic and liquid-liquid separations (high dose, Fig. 2).

(d) Cholinesterase Inhibition (i) In vitro Inhibition The percentage inhibition values produced by fraction (ii) (Fig. 3) and reference compounds are shown in Table 4. When the logarithm of the concentration of inhibitor was plotted against percentage inhibition a straight line was obtained which allowed calculation of I50 values. The I50 values for fraction (ii) and authentic oxygen analogue are in close agreement. The in vitro anticholinesterase activity of the oxygen analogue is shown to be 150 times greater than that of coumaphos . .. 630 W. J. ROULSTON, C. A. SCHUNTER, AND H. J. SCHNITZERLING

(ii) In vivo Inhibition The results of the in vivo cholinesterase inhibition experiment are shown in Figure 6. At the three coumaphos concentrations tested, cholinesterase inhibition was apparent before any of the larvae succumbed to treatment. In the later stages of the 0·002 and 0·0002% treatments a high inhibition level was associated with high larval mortality. However, 24 hr after the 0·0001 % treatment a 65% inhibition

100 1 ~

40 A

20 +

100 ~ o ;:: 80 ~ 0 60 • ::;: 0 40 z 0 20 + ~ :;: 0 ~ 20

100

80 o 60

20 + 0 • • • • --o----t:r~ 20 I o 4 9 10 11 12 13 24 46 HR AFTER DIPPING Fig. 6.-Percentage cholinesterase inhibition (0) and mortality (.) of larvae at various times after dipping in coumaphos suspensions of different concentrations: A, 0·002%; B, 0·0002%; 0, 0·0001%. was apparent with virtually no larval mortality, and 46 hr after dipping the larvae were as active as control larvae. The times for 50% inhibition of cholinesterase activity and the concentrations of oxygen analogue and coumaphos present at this level of inhibition are shown in Table 5.

IV. DISCUSSION The present investigations have shown that coumaphos penetrated rapidly into B. microplus larvae and was metabolized to solvent-soluble and water-soluble metabolites. One of these metabolites was identified as the oxygen analogue of METABOLISM OF COUMAPHOS IN CATTLE TICKS 631 coumaphos which was shown to be approximately 150 times more potent as a cholinesterase inhibitor than the parent compound. The activation of phosphoro. thioates in other biological systems has been described previously but, except in the case described by Brindley and Dahm (1964), the identification of the oxygen analogue has depended on chromatographic evidence. The need to positively identify the anticholinesterase metabolite was emphasized by the results of Hopkins and Knapp (1963) who showed that cattle grubs removed from cattle treated with fenchlorphos* contained an anticholinesterase metabolite which was not the oxygen analogue. In the present case the infrared and ultraviolet spectra of the anti· cholinesterase metabolite extracted from B. microplu8 identify it as the oxygen analogue of coumaphos. TABLE 5 CONCENTRATION OF COUMAPHOS AND OXYGEN ANALOGUE OF COUMAPHOS PRESENT AT TIMES OF 50% CHOLINESTERASE INHIBITION

Concentration (p.g/g) at Time of 50% Dipping Time (hr) for 50% Cholinesterase Inhibition Concentration Cholinesterase (%) Inhibition in vivo * Oxygen Coumaphost Analoguet

0·002 1·2 0·9 8·0

0·0002 2·5 0·2 0·6

0·0001 7·0 0·1 0·3 * Data obtained from Figure 6. t Data obtained from Figures 2 and 3 and other experiments not fully recorded in this paper.

It is now generally accepted that the toxicity of organic phosphorus compounds is due to their anticholinesterase properties (Chadwick and Hill 1947; Metcalf and March 1949; Mengle and Casida 1958; O'Brien 1960). In the in vivo inhibition experiment homogenization was carried out in the presence of substrate at relatively low temperatures to obviate the possible effect on the inhibition level of free inhibitor. The linear relation of reaction rate with time over the reaction period indicates that the values obtained were not affected by any free inhibitor, therefore the cholinesterase inhibition data (Fig. 6) are considered to be a true picture of in vivo conditions, showing that cholinesterase inhibition in larvae precedes death. If the amounts of coumaphos present at the times of 50% cholinesterase inhibition (Table 5) are divided by 150 (derived from the ratio of ho values in vitro for coumaphos and its oxygen analogue), the values which result are equal to the amounts of oxygen analogue equivalent in inhibition to coumaphos. These amounts are 0·05,0·004, and 0·00l ""gjg for the 0·002, 0·0002, and 0·000l % dipping con· centrations respectively. When compared with the amounts of oxygen analogue actually found these amounts are small and they would contribute only to a very small degree to the general level of in vivo cholinesterase inhibition. * Dimethyl 2,4,5-trichlorophenyl phosphorothionate. 632 W. J. ROULSTON, C. A. SCHUNTER, AND H. J. SCHNITZERLING

The water-soluble metabolites were tentatively identified by their RF values as two major metabolites (diethyl thiophosphoric and diethyl phosphoric acids) and three minor metabolites (the two desethyl derivatives of the two major metabolites and orthophosphoric acid). The neutralized reference diethyl thiophosphoric and phosphoric acids at 1 X 1O-3M failed to give any in vitro cholinesterase inhibition and their role in in vivo cholinesterase inhibition is considered negligible. Whereas the in vitro cholinesterase inhibition characteristics of the two minor metabolites were not determined by experiment, the possibility that these compounds might be inhibitors is extremely unlikely in view of their structural analogy with the non inhibiting diethyl acids. No cholinesterase inhibition or chromatography data are available concerning the hexane-soluble fraction (Figs. 1 and 2) as the activity was too low for any tests to be made. However, in view of the high hexane solubility of this fraction and the extremely small amount present, its significance in cholinesterase inhibition is considered negligible. Nothing is known about the identity or the .anticholinesterase activity of the metabolite with solvent-soluble and pol~u properties found only in larvae exposed to the lethal treatment of 0·002% coumaphos. It could be an intermediate metabolite which accumulates when hydrolytic systems cannot complete the degradation to the expected metabolites or it may result from a new metabolic mechanism which becomes predominant above a threshold dose. Until this intermediate is characterized, its role cannot be evaluated. In view of the high cholinesterase inhibition activity of the oxygen analogue together with the lack of any other potent anticholinesterase metabolite at the two lower dose levels, it is evident that the oxygen analogue is the metabolite of major biochemical significance involved in the toxic action of coumaphos in larval ticks. Even though the oxygen analogue is a more potent in vitro cholinesterase inhibitor than coumaphos these two compounds differ little in their toxicity either to B. microplu8 larvae or to adults (Stone, unpublished data). This agrees with the findings of Monroe and Robbins (1959) who reported that coumaphos and the oxygen analogue were equally toxic to houseflies by topical treatment although the oxygen analogue was 435 times more potent as an anticholinesterase agent in vitro. Differences in penetration rates and in hydrolytic stability in vivo may explain this anomaly.

V. ACKNOWLEDGMENTS Microanalyses were carried out by the Australian Microanalytical Service, Melbourne. VI. REFERENCES

ASPEREN, K. VAN (1960).-Toxic action of organophosphorus compounds p,nd esterase inhibition in houseflies. Biochem. Pharmac. 3: 136-46. BRINDLEY, W. A., and DAHM, P. A. (1964).-Identification of the in vitro anticholinesterase metabolite of methyl parathion. J. Econ. Ent. 57: 47-9. BRITISH STANDARD 1831 (1961).-"Recommended Common Names for Pesticides." (British Standards Institution: London.) CHADWICK, L. E., and HILL, D. L. (1947).-Inhibition of cholinesterase by di·isopropyl fluoro phosphate, physostigmine and hexaethyl tetraphosphate in the roach. J. N europhyaiol. 10: 235-46. METABOLISM OF COUMAPHOS IN CATTLE TICKS 633

CHAMBERLAIN, W. F., GATTERDAM, P. E., and HOPKINS, D. E. (1960).-Metabolism ofP32-Delnav in cattle. J. Econ. Ent. 53: 672-5. HOPKINS, T. L., and KNAPP, F. W. (1963).-Anticholinesterases in blood and cattle grubs from cattle treated with ronnel. J. Econ. Ent. 56: 872-4. KANE, P. F., COHEN, C. J., BETKER, W. R., and MACDOUGALL, D. (1960).-Assay of Co-Ral in technical material and formulated products. J. Agric. Food Chem. 8: 26-9. KAPLANIS, J. M., HOPKINS, D. E., and TREIBER, G. H. (1959).-Dermal and oral treatments of cattle with phosphorus-32 labelled Co-ral. J. Agric. Food Chem. 7: 483-6. MENGLE. D. C., and CASIDA, J. E. (1958).-Inhibition and recovery of brain cholinesterase activity in houseflies poisoned with organophosphate and carbamate compounds. J. Econ. Ent. 51: 750-7. METCALF, R. L., and MARCH, R. B. (1949).-Studies of the mode of action of parathion and its derivatives and their toxicity to insects. J. Econ. Ent. 42: 721-8. MONROE, R. E., and ROBBINS, W. E. (1959).-Studies on the mode of action of synergized Bayer 21/199 and its corresponding phosphate in the housefly. J. Econ. Ent. 52: 643-7. O'BRIEN, R. D. (1960).-"Toxic Phosphorus Esters." (Academic Press, Inc.: New York.) PLAPP, F. W., and CASIDA, J. E. (1958).-Ion exchange chromatography for hydrolysis products of organophosphate insecticides. Analyt. Chem. 30: 1622-4. RADELEFF, R. D., and WOODARD, G. T. (1956).-Cholinesterase activity of normal blood of cattle and sheep. Vet. Med. 51: 512-14. ROULSTON, W. J., and WILSON, J. T. (1965).-Chemical control of the cattle tick Boophilus microplus. Bull. Ent. Res. 55: 617-35. SCHNITZERLING, H. J., and SCHUNTNER, C. A. (1965).-Column chromatography of acid esters of phosphoric acids in a non-aqueous system. J. Chromatogr. 20: 621-3. SHAW, R. D., and MALCOLM, H. A. (1964).-Resistance of Boophilus microplus to organophosphorus insecticides. Vet. Rec. 76: 210-11.